Receptor tyrosine kinase agonist, composition for cell culture medium, and undifferentiated maintenance composition

Abstract

A receptor tyrosine kinase agonist which is a single-domain antibody having a cell growth activity, and is at least one receptor tyrosine kinase agonist selected from the group consisting of a human fibroblast growth factor receptor 1 (FGFR1), a human fibroblast growth factor receptor 2 (FGFR2), a human fibroblast growth factor receptor 3 (FGFR3), and a human fibroblast growth factor receptor 4 (FGFR4).

1 . A receptor tyrosine kinase agonist comprising a single-domain antibody having a cell growth activity,
 wherein the receptor tyrosine kinase agonist is   at least one receptor tyrosine kinase agonist selected from the group consisting of a human fibroblast growth factor receptor 1 (FGFR1), a human fibroblast growth factor receptor 2 (FGFR2), a human fibroblast growth factor receptor 3 (FGFR3), and a human fibroblast growth factor receptor 4 (FGFR4).   
     
     
         2 . The receptor tyrosine kinase agonist according to  claim 1 ,
 wherein an EC 50  value of the cell growth activity is 10 μg/mL or less.   
     
     
         3 . The receptor tyrosine kinase agonist according to  claim 1 ,
 wherein the receptor tyrosine kinase agonist is a dimer in which the single-domain antibodies having the same sequence are linked by a linker.   
     
     
         4 . The receptor tyrosine kinase agonist according to  claim 3 ,
 wherein the linker is an oligopeptide linker or a chemical linker.   
     
     
         5 . The receptor tyrosine kinase agonist according to  claim 1 ,
 wherein a denaturation temperature (Td) is 65° C. or higher.   
     
     
         6 . The receptor tyrosine kinase agonist according to  claim 1 ,
 wherein a value of an equilibrium dissociation constant (KD) is 5×10 −9  M or less.   
     
     
         7 . The receptor tyrosine kinase agonist according to  claim 1 ,
 wherein an amino acid sequence of a complementarity determining region (CDR) of the single-domain antibody includes an amino acid sequence represented by SEQ ID NO: 29 or an amino acid sequence represented by SEQ ID NO: 30.   
     
     
         8 . The receptor tyrosine kinase agonist according to  claim 1 ,
 wherein the single-domain antibody includes an amino acid having characteristics of any one of the following (1) to (5) at a position defined (specified) according to Kabat numbering:
 (1) a 37 th  amino acid is any amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), and valine (V), 
 (2) a 41 st  amino acid is proline (P), 
 (3) a 44 th  amino acid is any amino acid selected from the group consisting of glutamine (Q), glycine (G), glutamic acid (E), and lysine (K), 
 (4) a 45 th  amino acid is arginine (R) or leucine (L), and 
 (5) a 47 th  amino acid is any amino acid selected from the group consisting of leucine (L), alanine (A), tryptophan (W), glycine (G), and phenylalanine (F). 
   
     
     
         9 . A medium composition for cell culture, containing 0.1 ng/ml to 10 μg/mL of a receptor tyrosine kinase agonist,
 wherein the receptor tyrosine kinase agonist includes a single-domain antibody having a cell growth activity, and 
 is at least one selected from the group consisting of a human fibroblast growth factor receptor 1 (FGFR1), a human fibroblast growth factor receptor 2 (FGFR2), a human fibroblast growth factor receptor 3 (FGFR3), and a human fibroblast growth factor receptor 4 (FGFR4). 
 
     
     
         10 . The medium composition for cell culture according to  claim 9 ,
 wherein a cell grown by the cell growth activity is at least one cell selected from the group consisting of a fibroblast, a mesenchymal stem cell, and an iPS cell.   
     
     
         11 . A composition for maintaining an undifferentiated state of a stem cell, the composition comprising the receptor tyrosine kinase agonist according to  claim 1  as an active ingredient. 
     
     
         12 . The composition for maintaining an undifferentiated state according to  claim 11 ,
 wherein the stem cell is a mesenchymal stem cell or an iPS cell.   
     
     
         13 . The receptor tyrosine kinase agonist according to  claim 2 ,
 wherein the receptor tyrosine kinase agonist is a dimer in which the single-domain antibodies having the same sequence are linked by a linker.   
     
     
         14 . The receptor tyrosine kinase agonist according to  claim 13 ,
 wherein the linker is an oligopeptide linker or a chemical linker.   
     
     
         15 . The receptor tyrosine kinase agonist according to  claim 2 ,
 wherein the single-domain antibody includes an amino acid having characteristics of any one of the following (1) to (5) at a position defined (specified) according to Kabat numbering:
 (1) a 37 th  amino acid is any amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), and valine (V), 
 (2) a 41 st  amino acid is proline (P), 
 (3) a 44 th  amino acid is any amino acid selected from the group consisting of glutamine (Q), glycine (G), glutamic acid (E), and lysine (K), 
 (4) a 45 th  amino acid is arginine (R) or leucine (L), and 
 (5) a 47 th  amino acid is any amino acid selected from the group consisting of leucine (L), alanine (A), tryptophan (W), glycine (G), and phenylalanine (F).